Tropoelastin is the polypeptide precursor of elastin, a cross-linked fibrous protein essential to the structure and function of lung, large blood vessels and other tissues. In fetal bovine nuchal ligament, at least three distinct tropoelastin isoforms are produced, each translated from separate mRNAs which, in turn, are transcribed from a single gene. This proposal is designed to characterize the different tropoelastin isoforms produced by elastic tissues and to study the developmental regulation of their expression. Tropoelastin isoforms produced by bovine nuchal ligaments will be examined from immunoprecipitates of cell-free translated mRNA and compared with the pattern of isoforms isolated from ligament tissue. S-1 nuclease mapping will be done to determine which exons of the elastin gene are spliced from isoform-specific transcripts. Findings from studies on nuchal ligament will be compare with similarly derived observations from other elastic tissues, such as aorta, lung and skin, and from elastin-producing cells in culture. The cellular data will provide a basis for further studies on aberrant tropoelastin expression in cells under experimental manipulation or derived from patients with elastin-associated disease. The role of the different tropoelastin isoforms in elastin fiber formation will be addressed. Antibodies will be raised against synthetic peptides specific for individual isoforms, as defined by S-1 nuclease mapping, and used to affect fiber formation by fetal bovine chondrocyts in vitro. Additional studies will be done to determine what mechanisms are involved in regulating tropoelastin expression. Parameters to be examined include, mRNA transcription and turnover rates, levels of functional and steady-state tropoelastin mRNA, translational efficiency and protein half-life. The distribution of tropoelastin mRNA expression in elastic tissues will be determined by in situ hybridization using isoform-specific oligomeric probes. Unlike many other connective tissue components, essentially all elastin synthesis occurs only during fetal and neonatal periods, however production is reinitiated in some disease states such as pulmonary hypertension and emphysema. Therefore, understanding the full complexity of elastin production will add to our knowledge of how this important matrix component is assembled and functions.